Inhibition of Chemokine CCL7 or Receptor CCR3 of Same for the Treatment and Diagnosis of Prostate Cancer

ABSTRACT

The invention concerns an inhibitor of the expression of chemokine CCL7 or an inhibitor of the expression of the receptor CCR3 or an inhibitor of CCL7/CCR3 interaction for the use of same to prevent or treat the extension of prostate cancer outside the prostatic capsule in a subject. The invention also concerns a method for determining the degree of aggressiveness of a prostate cancer tumour in a subject suffering from prostate cancer, comprising a step of determining the concentration or level of expression of the receptor CCR3 in a sample of prostate tumour cells obtained from said subject.

The present invention relates to the field of the diagnosis andmedicinal products intended for the treatment of prostate cancer.

Prostate cancer is the cancer most frequently observed in men over 50years of age and represents the second cause of cancer-related death indeveloped countries. As for all solid cancers, the extension of theprostate cancer is based on the TNM classification (T for tumor, N for“nodes” or lymph node involvement, M for metastasis) (Ohori, Wheeler etal. 1994; Salomon, Azria et al. 2010).

Prostate cancers are thus classified in four stages:

1) localized prostate cancer: T1 [tumor not palpable or not visible withimaging] and T2 [tumor limited to the prostate (including apex andcapsule)], N0, M0;

2) locally advanced prostate cancer: T3 [extension beyond prostaticcapsule] and T4 [Extension to adjacent organs (neck of bladder, urethralsphincter, rectum, pelvic wall) or tumor attached to pelvic wall], N0,M0;

3) prostate cancer with lymph node involvement, all T, N1, M0; and

4) metastatic prostate cancer: all T, all N, M1.

The prostate is surrounded by adipose tissue (periprostatic adiposetissue or PPAT). The presence of the tumor in periprostatic adiposetissue thus represents the first stage of local extension of prostatecancer (stage pT3a) and is an established criterion of poor prognosis(Magi-Galluzzi, Evans et al. 2011; Houimel and Mazzucchelli 2013). Theinvasion of the surrounding tissues may be followed by remotedissemination of cancer cell (metastasis). These metastases areessentially localized in the bone and are the cause of the majority ofdisease-related deaths (mortality rate of approximately 90%). The bones,essentially consisting of bone tissue, also contain hematopoietictissue, adipose tissue, vessels, nerves, cartilaginous tissue andconnective tissue. As such, adipose tissue is present both at the localand remote dissemination stages of prostate cancer.

Among the cells making up adipose tissue, particularly periprostaticadipose tissue, mature adipocytes are mainly found along with, in aso-called “stromal vascular” fraction, other cells such as progenitorcells (Adipose Derived Stem Cells [ADSCs] and preadipocytes),fibroblasts, macrophages, lymphocytes, pericytes and endothelial cells(Ouchi, Parker et al. 2011). Adipocytes have a role in the energyfluctuations induced by food intake or fasting states by storing energyin the form of triglycerides or by releasing same in the form of fattyacids (FAs). Adipocytes are also active endocrine cells which secrete agreat variety of molecules (called adipokines), which are particularlyinvolved in appetite and energy balance regulation, the lipidmetabolism, insulin sensitivity and blood pressure regulation (Ouchi,Parker et al. 2011). Adipocyte secretions include growth factors,chemokines, pro-angiogenic molecules or pro-inflammatory molecules(Ouchi, Parker et al. 2011). Tourniaire, Romier-Crouzet et al. (2013)reported that, under obesity conditions, the secretory profile ofadipocytes is modified, and the secretion of certain chemokines may beincreased. Furthermore, as summarized in Allott, Masko et al. (2013), aconvincing set of epidemiological data demonstrates that obesity is afactor of poor prognosis in prostate cancer with an increase in localand remote dissemination. Finally, independently of obesity, recent datademonstrate that the extent of the PPAT is positively associated withthe aggressiveness of prostate cancer (Kiss, Longden et al. 2009).

Chemokines represent a large group of small proteins (of approximately 8to 11 kDa) which interact with receptors belonging to the superfamily ofG-protein-coupled seven-transmembrane domains receptors (GPCRs). Most ofthese have four characteristic cysteine residues which form twodisulfide bridges, one between the first and the third cysteine residueand one between the second and fourth cysteine residue. According to thenumber of amino acids present between the first two cysteines (situatedin the N-terminal part), chemokines are classified into four groups: C,CC, CXC and CX3C. Chemokines have various functions, the most studiedwhereof is attraction (chemotaxis) and immune system cell activationstate control. In cancer, these chemokines and the receptors thereof areknown for regulating the tumoral process (proliferation, migration,invasion, angiogenesis, leukocyte infiltration). Some of these areproduced by the tumor cells themselves, but also by stromal cells suchas cancer-associated fibroblasts (CAFs), endothelial cells or immunecells (macrophages, lymphocytes) (Balkwill 2004).

Of the chemokine receptors potentially involved in cancer, the receptorCXCR4 is currently considered to be one of the most important receptors.The interaction of CXCR4 with the ligand CXCL12 thereof plays a role inthe cellular migration of many cancers, including the prostate, inparticularly in the context of bone metastasis (Taichman, Cooper et al.2002). In vitro studies have demonstrated that CXCL12 stimulates theinvasive nature of prostate tumor cells and that it can be blocked usinga pharmacological inhibitor (for example the CXCR4 antagonist calledAMD3100) or a blocking antibody. The interaction between the receptorCCR2 and the chemokine CCL2 has also been identified for the involvementthereof in prostate tumor cell migration (Loberg, Day et al. 2010).Furthermore, the expression of the receptor CCR2 is correlated with theaggressiveness of prostate tumors (Lu, Cai et al. 2007). Furtherchemokine receptors such as CXCR1 and CXCR2 have been described asplaying a role in the invasion and growth of prostate tumor cells viathe chemokines CXCL1, CXCL5 or CXCL8 (Inoue, Slaton et al. 2000; Begley,Kasina et al. 2008).

Further chemokine receptors liable to interact in the migration of tumorcells in other types of cancers have also been identified, such as forexample the receptors CCR1 (GeneID: 1230) (Loetscher, Pellegrino et al.2001) and CCR3 (GeneID: 1232) (Loetscher, Pellegrino et al. 2001; Jung,Che et al. 2010; Johrer, Zelle-Rieser et al. 2005; Lee, Kim et al.2010). The latter are very strongly expressed by eosinophils orbasophiles and less significantly by lymphocytes (Th1 and Th2) and someepithelial cells. CCR1 and CCR3 are also involved in eosinophilmigration regulation and play a major role in numerous inflammatorydiseases such as asthma (Joubert, Jajoie-Kadoch et al. 2008). To date, anumber of ligands of CCR3 have been identified: CCL5, CCL7, CCL11,CCL13, CCL15, CCL24, CCL26 and CCL28. Jung, Che et al. (2010)demonstrated in carcinomas of the upper airways that CCR3 may play arole in cell migration via the chemokine CCL7 (GeneID:6354; Kuri-Harcuchand Green 1978). It has been demonstrated that the receptor CCR3 isoverexpressed in kidney tumors (Jung, Che et al. 2010) and melanoma(Lee, Kim et al. 2010). Very recently, a preliminary study relating to asingle prostate cancer line Du-145 demonstrates that the receptor CCR3promotes, via CCL11 (a different chemokine from CCL7), in vitromigration and invasion of cancer cells. In this study, the authors didnot focus on the chemokines secreted by adipose tissue (Zhu, Liao et al.2013). Furthermore, the in vivo effect of the inhibition of the receptorCCR3 and the expression thereof in human prostate tumors were notaddressed in this study.

In all these studies relating to the role of chemokines in prostatecancer progression, the role of PPAT secretions was not envisaged, norwas the link with obesity and/or the extent of the periprostatic adiposetissue mentioned.

The therapeutic strategy for localized tumors is currently based on theD'Amico classification if the characteristics of the cancer areconsidered exclusively (Guide ALD30, HAS/INCA, 2012). Indeed, furtherfactors such as the characteristics of the patient and his/herpreferences (in particular in the case where therapeutic alternativeshaving an equivalent benefit/risk ratio exist) are also considered. TheD'Amico classification (Agrawal, Maxwell et al. 2009; Ohori, Wheeler etal. 1994) makes it possible to account for the following criteria: thesize of the tumor, the Gleason score (Kiss, Longden et al. 2009) and theserum marker PSA (Prostate-Specific Antigen). The Gleason classificationis based on the degree of differentiation of the tumor, scored fromgrade 1 (differentiated) to 5 (very poorly differentiated). This scoreis the sum of the two grades most frequently represented in the tumoranalyzed. It varies from 2 to 10. The score of 2 corresponds to a tumorvery similar to benign tissue. The higher the score, the more aggressivethe tumor. The D'Amico classification defines three subgroups oflocalized prostate cancer according to the risk of recurrence (referringto the risk of biological recurrence 10 years after a local treatment),i.e. three cancer progression risk levels: a low risk, an intermediaterisk and a high risk. The three subgroups defined according to theclinical and biological characteristics thereof are:

1) low-risk localized prostate cancer: TNM≤T2a (involvement of half ofone lobe or less) and Gleason Score≤6 and PSA value (ng/ml)≤10;

2) intermediate-risk prostate cancer: TNM≤T2b (involvement of more thanhalf of one lobe without involvement of the other lobe) and GleasonScore=7 and PSA value (ng/ml)=10-20; and

3) high-risk localized prostate cancer: TNM≥T2c (involvement of bothlobes) and Gleason Score≥8 and PSA value (ng/ml)>20.

There is currently a need to develop treatments suitable for delaying orpreventing the local and remote dissemination stages in order to make itpossible not only to prevent the development of metastatic disease butalso to maintain the prostate cancer in a potentially curable form foras long as possible.

There is also a need to identify using a diagnostic method localizedprostate cancer tumors having a high aggressive potential in order totreat same suitably.

Within the scope of their research work, the inventors characterized aspecific regulation pathway of the local dissemination process ofprostate cancer via the involvement of the chemokine receptor CCR3 underthe dependency of one of these ligands, CCL7, expressed by PPAT.

The inventors demonstrated in vitro that a conditioned medium frommature adipocytes (CM-Ad) obtained from the differentiation of the mousepre-adipocyte line F442A stimulates the migration of human and mouseprostate cells. This migration is inhibited by the use of a knownCCR1/CCR3 inhibitor (UCB35625) in all the prostate cancer cell linesstudied whereas CCR1 is not expressed in these lines. The importance ofCCR3 in the MC-Ad-induced migration of cancerous prostate cells wasconfirmed by the absence of effect of UCB35625 on the migration ofdifferent tumor lines representative of different cancers (breast,colon, pancreas, melanoma), apart from a slight effect on the migrationof the aggressive breast cancer line MDA-MB 231. Inhibition was alsoobserved for blocking antibodies targeted against CCR3 in all the modelsstudied and when CCR3 expression is invalidated. In MC-Ad, a single CCR3ligand was detected: CCL7. The inhibition of this ligand by a blockingantibody inhibits the migration of human and mouse prostatic cellsagainst CM-Ad. This ligand is also present in the conditioned mediumfrom mouse perigonadal and human periprostatic adipose tissue and theconditioned medium from primary adipocytes isolated from mouseperigonadal adipose tissue. The capability of inducing the migration ofprostate tumor cells along a CCR3/CCL7 pathway was also detected forconditioned medium from mouse perigonadal and human periprostaticadipose tissue and the conditioned medium from primary adipocytesisolated from mouse perigonadal adipose tissue.

The inventors also demonstrated in vivo that intraprostatic injection oftumor lines invalidated for CCR3 results in a reduction in tumoral massalong with a lack of disappearance of periprostatic adipose tissue underthe effect of tumoral infiltration. Furthermore, the inventorsdemonstrated that the receptor CCR3 is expressed in human tumors andthat this expression is linked with the aggressiveness of the tumors(correlation with the Gleason score, the percentage ofnon-differentiated cells, the peripheral site of the tumor) and with theextra-prostatic dissemination thereof (TNM stage). The expression is ofthe receptor CCR3 is also correlated with biological recurrence andsurgical treatment failure. Finally, medullary adipocytes are capable ofsecreting CCL7 and the conditioned medium from these adipocytes inducesthe migration of prostate tumor cells, said migration being inhibited byUCB35625 or a blocking antibody targeted against CCR3 and/or CCL7. Theseresults were obtained with mouse medullary adipocytes (obtained from theex vivo differentiation of adipocyte progenitors) and human medullaryadipocytes (isolated directly from human yellow marrow). All these datademonstrate that the CCR3/CCL7 pathway appears to be involved in remotedissemination (bone metastasis) of prostate cancer via the ability ofmedullary adipocytes to secrete CCL7. These data also demonstrate theinterest of inhibiting CCR3 or CCL7 in prostate cancer with a view toinhibiting local or remote extension.

Furthermore, this strategy aimed at inhibiting CCR3 or CCL7 is ofparticular interest for obese subjects or subjects with abundant PPAT(the surface area whereof is greater than or equal to 10 cm²). Indeed,the inventors demonstrated that the genic expression of CCL7 isincreased in the adipose tissues of obese mice and subjects. Thesecretion thereof is increased in conditioned medium from perigonadaltissue from obese mice and conditioned medium from primary adipocytesisolated from perigonadal tissue from obese mice. The migration of humanprostatic cancer cells against conditioned medium from perigonadaltissue from obese mice and conditioned medium from primary adipocytesisolated from perigonadal tissue from obese mice is increased withrespect to the same media obtained with animals of normal weight. Thisincrease is completely inhibited by UCB35625 or blocking antibodiestargeted against CCR3 or CCL7. In vivo, the increase in tumoral massobserved under obesity conditions is no longer present when the receptorof CCR3 is invalidated. In humans, expression of the receptor CCR3 issignificantly increased in obese subjects. Furthermore, there is acorrelation between the extent of the PPAT viewed using nuclear magneticresonance imaging (MRI) or tomodensitometry and CCR3 expression.Finally, in humans, the medullary adipocytes obtained from obesesubjects have an increased capability of inducing the migration ofprostate tumor cells and this effect is inhibited by UCB35625.

The present invention consequently relates to an inhibitor of theexpression of the chemokine CCL7 or an inhibitor of the expression ofthe receptor CCR3 or an inhibitor of the expression of the receptor CCR3or an inhibitor of CCL7/CCR3 interaction for the use thereof to preventor treat the extension of prostate cancer outside the prostatic capsulein a subject.

The term “extension of prostate cancer outside the prostatic capsule”denotes locally advanced prostate cancer (as per the TNM classification:T3 or T4, N0, M0), prostate cancer with pelvic lymph node involvement(as per the TNM classification: all T, N1, M0) or metastatic prostatecancer (as per the TNM classification: all T, all N, M1).

According to one preferred embodiment of the invention, said inhibitoris used in an adult male who is obese or has abundant periprostaticadipose tissue.

Obesity is a state characterized by an excess of adipose mass. The BodyMass Index (BMI) is an international standard for measuring overweightand obesity. It is defined as the weight divided by the height squared,expressed in kg/m². A subject is considered to be obese when this valueis greater than or equal to 30 kg/m².

The term abundant periprostatic adipose tissue (PPAT) denotes a PPATwherein the surface area is greater than or equal to 10 cm².Advantageously, the PPAT surface area is measured using a method formeasuring and calculating the area of an ellipse using images, obtainedfor example by CT or MRI imaging on said subject, of cross-sections atthe femur at the point where the fusion of the symphysis pubis starts.The periprostatic adipose tissue deposit for which an image is obtainedis situated between the anterior part of the prostate and the symphysispubis. Such a method is described in the Materials and Methods sectionhereinafter.

The limit of 10 cm² was defined statistically as being the surface areabeyond which all patients have a significantly more aggressive cancer(Gleason score greater than or equal to 7).

The inhibitors of the expression of CCL7 or CCR3 include nucleic acidssuch as optionally modified antisense oligonucleotides, interfering RNA,small hairpin RNA (shRNA or miRNA) or ribozymes, targeting the geneencoding CCL7 or the gene encoding CCR3. Such inhibitors are describedby Zhu, Liao et al. 2013; Agrawal, Maxwell et al. 2009; Zhu, Liu et al.2014.

According to one preferred embodiment, the inhibitor of the expressionof CCR3 is a shRNA targeting the gene CCR3. It is advantageously chosenfrom the shRNA: m4CCR3 (SEQ ID NO: 1), m5CCR3 (SEQ ID NO: 2), m6CCR3(SEQ ID NO: 3).

According to one preferred embodiment, the inhibitor of the expressionof CCR3 is an antisense oligonucleotide, optionally modified, targetingthe gene CCR3. It is advantageously chosen in the group consisting ofthe antisense oligonucleotides TOP004 having the sequence SEQ ID NO: 14,TOP005 having the sequence SEQ ID NO: 15, TOP030 having the sequence SEQID NO: 16, TOP030-P²M-7-DAP having the sequence SEQ ID NO: 17,TOP030-P²M-8-DAP having the sequence SEQ ID NO: 18, TOP030-P²M-9-DAPhaving the sequence SEQ ID NO: 19, TOP030-P²M-10-DAP having the sequenceSEQ ID NO: 20, TOP030-P²M-11-DAP having the sequence SEQ ID NO: 21,TOP030-P²M-12-DAP having the sequence SEQ ID NO: 22, TOP030-P²M-13-DAPhaving the sequence SEQ ID NO: 23, TOP030-P²M-14-DAP having the sequenceSEQ ID NO: 24 and TOP030-P²M-15-DAP having the sequence SEQ ID NO: 25.

The term “an inhibitor of CCL7/CCR3 interaction” denotes a compoundcapable of inhibiting the activation of CCR3 by CCL7, without activatingCCR3 itself. An inhibitor of CCL7-CCR3 interaction includes compoundscapable of interacting with CCL7 and inhibiting the bonding thereof withCCR3 or inhibiting the activation of CCR3 resulting from said bonding,along with compounds capable of interacting with CCR3, and inhibitingthe bonding thereof with CCL7 or the activation thereof resulting fromsaid bonding. In particular, an inhibitor of CCL7/CCR3 interactionincludes competitive and non-competitive CCR3 antagonists (referred toas CCR3 antagonists), preferably competitive CCR3 antagonists.

The inhibitors of CCL7/CCR3 interaction include organic molecules. Byway of example of organic molecules inhibiting CCL7/CCR3 interaction,mention may be made of the compounds having formulas I to DCCLXXXIVdescribed hereinafter, preferably the compound UCB356625(C₃₀H₃₇Cl₂IN₂O₂) having formula DLI (hereinafter).

By way of examples of organic molecules inhibiting CCL7/CCR3interaction, mention may also be made of irreversible CCR3 inhibitorssuch as the compounds morpholine-acetamide and morpholine urea, inparticular those described in the Applications WO 2002/26723 and WO03/082293, and the U.S. Pat. Nos. 7,101,882, 7,157,457, 7,531,651 and7,560,548.

The inhibitors of CCL7/CCR3 interaction also include peptides. By way ofexample of peptides inhibiting CCL7/CCR3 interaction, mention may bemade of the peptide met-RANTES (Kiss, Longden et al. 2009), or furtherpeptides described in Houimel and Mazzucchelli 2013; Elsner, Escher etal. 2004; Kiss, Longden et al. 2009; Elsner, Petering et al. 1997;Loetscher, Pellegrino et al. 2001 and International ApplicationW02000073327.

The inhibitors of CCL7/CCR3 interaction also include antibodies targetedagainst CCR3 or CCL7, preferably blocking antibodies, which may bepolyclonal or monoclonal.

The term “antibody” also includes functional antibody fragments,including chimeric, humanized, single-chain antibody fragments orfragments thereof (e.g. fragments Fv, Fab, Fab′ and F(ab′)₂). Preferredantibodies are those which are targeted against CCL7 or CCR3.Advantageously, the antibody is a monoclonal antibody or a fragmentthereof. The methods for preparing monoclonal antibodies are well-knownto those skilled in the art. The monoclonal antibodies may be preparedby immunizing a mammal (for example, a mouse, a rat, a rabbit, a camelidor humans) with the chemokine CCL7 or the receptor CCR3 or purifiedfragments thereof. By way of example of antibodies inhibiting CCL7/CCR3interaction and by way of example anti-CCR3 monoclonal blocking antibody(reference D083-3, clone 444-11, MBL international, Houimel andMazzucchelli 2013) or anti-CCL7 polyclonal blocking antibody (referenceAF-282-NA, R&D systems, Szymczak and Deepe 2009).

The inhibitors of CCL7/CCR3 interaction also include peptidomimetics.

The term “peptidomimetic” denotes a molecule which is not a peptide butwhich imitates aspects of the structure of peptides (Elsner, Petering etal. 1997). By way of example of peptidomimetics inhibiting CCL7/CCR3interaction, mention may be made of those described in the U.S. Pat. No.7,488,717.

The inhibitors of CCL7/CCR3 interaction also include aptamersrecognizing CCL7 or CCR3. Aptamers are molecules with a single strand ofnucleic acid (DNA or RNA) which are selected for the ability thereof tobind with a target molecule.

Further inhibitors of CCL7/CCR3 interaction may be identified, forexample, by screening a collection of candidate compounds for theability thereof to inhibit the activation of CCR3 in the presence ofCCL7. Methods for measuring the activation of RCPG receptors which areknown per se, and which are for example used routinely in high-speedscreening tests, may be used to evaluate the activation of CCR3. By wayof example of methods for identifying inhibitors of CCL7/CCR3interaction, mention may be made of in vitro ligand/receptor interactiontests (Loetscher, Pellegrino et al. 2001), time-resolved fluorimetricassays (Kuri-Harcuch and Green 1978) or surface Plasmon resonancescreening (Ohori, Iacono et al. 1994).

The inhibitors used according to the invention may be administered to asubject alone, or in a mixture with at least one pharmaceuticallyacceptable excipient which may be any excipient known to those skilledin the art. The pharmaceutically acceptable excipients vary according tothe inhibitor used and the method of administration chosen.

The methods and routes of administration of the inhibitors usedaccording to the invention may be adapted by those skilled in the artaccording to the subject and the inhibitor used. By way of example, theinhibitors may be formulated for administration by the oral or nasalroute, or by injection by the intravenous, intramuscular or subcutaneousroute.

The determination of the dose at which said inhibitor is used accordingto the invention may be performed using techniques known to thoseskilled in the art, for example during clinical trials. This dose willbe dependent on various factors comprising in particular the activity ofthe inhibitor, the method of administration, the duration ofadministration, the duration of the treatment, other medicinal productsor compounds used in conjunction with the inhibitor, age, sex, weight,general health and previous medical history of the subject treated.

The present invention also relates to a method for preventing ortreating the extension of prostate cancer outside the prostatic capsule,comprising the administration of an effective quantity of at least oneinhibitor of the expression of the chemokine CCL7 or an inhibitor of theexpression of the receptor CCR3 or an inhibitor of CCL7/CCR3interaction, to a subject requiring this treatment. Obviously, thespecifications described above also apply to this aspect of theinvention.

The term effective quantity denotes a quantity of inhibitor usedaccording to the invention to produce the biological result sought.

The present invention also relates to an ex vivo method for determiningthe degree of aggressiveness of a prostate cancer tumor in a subjectsuffering from prostate cancer, or for determining the risk ofbiological recurrence of prostate cancer in a subject, comprising thefollowing steps:

a) determining the concentration or level of expression of the receptorCCR3 in a sample of prostate tumor cells obtained from said subject,

b) comparing the concentration or level of expression of the receptorCCR3 determined in step a) with the reference concentration or level ofexpression of the receptor CCR3 in healthy prostate epithelium,

a concentration or a level of expression of the receptor CCR3, in saidsample of prostate tumor cells from said subject, greater than saidreference concentration or level of expression being the indication ofan aggressive prostate cancer tumor with a high extraprostaticdissemination potential or of a risk of biological recurrence ofprostate cancer.

A sample of prostate tumor cells may be obtained according to methodsknown to those skilled in the art. For example, prostate biopsies,optionally targeted with a radiological examination, are the standardexamination for establishing the diagnosis of prostate cancer (Ohori,Wheeler et al. 1994). These samples may also be obtained from endoscopicresection of the prostate or from total prostate resection specimens(Ohori, Wheeler et al. 1994).

A sample of healthy prostate epithelium may be obtained according tomethods known to those skilled in the art. For example, this may consistof normal prostate epithelium present outside the cancer sites inprostate biopsies, healthy prostate tissue present next to cancer siteson specimens from endoscopic resection and/or total prostate resection(Ohori, Wheeler et al. 1994).

The term “aggressive prostate cancer tumor with high extraprostaticdissemination potential” denotes localized cancers wherein theprogression is marked rapidly by local dissemination. These cancers at ahigh risk of progression have been defined using the D'Amicoclassification, as tumors having a TNM≥T2c (involvement of both lobes)or a Gleason score≥8 or PSA value (ng/ml)>20.

Said subject is preferably an adult male who is obese and/or hasabundant PPAT.

The measurement of the concentration of the receptor CCR3 in a sample ofcells (prostate cells or prostate epithelium cells) may be performedusing a suitable immunological method (e.g. immunofluorescence,immunohistochemistry) by means of at least one specific antibody for thereceptor CCR3.

The specific antibody for the receptor CCR3 may be polyclonal ormonoclonal. It may be of human or non-human origin (for example mouse),humanized, chimeric. It may be recombinant or synthetic. It may also bean antibody fragment (for example Fab′₂ or Fab fragments) comprising adomain of the initial antibody recognizing the target antigen of saidinitial antibody. This may be for example the clone Y31, (AbcamCambridge, Mass., USA; Lee, Kim et al. 2010).

The reference concentration of the receptor CCR3 in the healthy prostateepithelium is dependent on the method used for measuring theconcentration.

By way of example, the reference concentration of the receptor CCR3 inthe healthy prostate epithelium may be obtained by immunohistochemicalanalysis of prostate epithelium. The immunolabeling may be the subjectof a semi-quantitative visual evaluation. Four intensity levels may bedistinguished: no labeling (0), weak labeling (1+), intermediatelabeling (1++) and strong labeling (1+++), as described in thepublications (Ohori and Scardino 1994) and (Saitoh, Miura et al. 1994).The reference concentration of the receptor CCR3 in the healthy prostateepithelium is then between zero and 1+.

The method according to the present invention may comprise an additionalstep (c) after the comparison step (b), comprising the classification ofsaid subject suffering from prostate cancer according to the degree ofaggressiveness of the prostate cancer tumor, based on the level ofconcentration or expression of the receptor CCR3.

The present invention also relates to the use of a specific antibody forthe receptor CCR3, as defined above, for determining the degree ofaggressiveness of a prostate cancer tumor in a subject suffering fromprostate cancer.

Besides the preceding arrangements, the invention comprises furthernon-limiting arrangements, which will emerge from the experimentalexamples hereinafter, along with the appended figures:

FIG. 1: Identification of chemokine receptors and role in the migrationof prostate tumor cells. (A) Migration of LNCaP,

C4-2B, Du-145 and PC-3 human prostate tumor cells against serum-freemedium, medium containing 10% serum, or conditioned medium from matureadipocytes (CM-Ad). (B) Expression of the chemokine receptors CXCR1,CXCR2, CXCR4, CCR1, CCR2 and CCR3 by flow cytometry in Du-145 and PC-3cells. (C) Migration of PC-3 against conditioned medium of adipocytes(CM-Ad) in the presence of CXCR4 inhibitor (AMD3100), CXCR1 and CXCR2inhibitor (SB25002), CCR2 inhibitor (SB202525), CCR1 and CCR3 inhibitor(UCB35625) and 10 μg/ml of control IgG1, anti-CCR1 or anti-CCR3antibody. (D) Expression of the receptor CCR3 by flow cytometry in humanprostate (Du-145 and PC-3), breast (T47D and MDA-MB231), melanoma(501Mel and Lu1205), pancreas (CAPAN and PANC-1) and colon (sw620 andsw480) tumor cells) against conditioned medium from mature adipocytes(CM-Ad) in the presence of CCR1 and CCR3 inhibitor (UCB35625).

FIG. 2: Identification of adipocyte chemokines and role in the migrationof prostate tumor cells. (A) analysis of adipocyte secretome (adipocytes3T3-F442A) by mass spectrometry and identification of adipocytechemokines. (B) Migration of Du-145 and PC-3 cells against medium(serum-free) optionally supplemented with the human recombinantchemokine CCL7 (1-100 ng/ml). Migration of Du-145 and PC-3 cells againstCM-Ad after treatment with anti-CCL7, CCR1 and CCR3 blocking antibodies.(C) Analysis of secretion of CCL7 in conditioned media of mouseperigonadal (AT-mu-PG) and human periprostatic (AT-hu-PP) adiposetissues by ELISA. Migration of the cells PC-3 against conditioned mediumfrom mouse perigonadal (AT-mu-PG) and human periprostatic (AT-hu-PP)adipose tissues after treatment with CCR1/CCR3 inhibitor (200 nMUCB35625) or with 10 μg/ml of control IgG1, anti-CCR3 or anti-CCL7antibody.

FIG. 3: The expression of the chemokine CCL7 is positively regulated inthe adipose tissue and adipocytes of obese subjects. (A) Analysis of themRNA of CCL7 by qPCR in human visceral adipose tissue of normal-weightor obese patients. Analysis of the mRNA of CCL7 by qPCR in perigonadaladipose tissue of normal-weight mice, on a high-fat diet, and of obeseDb/Db mice. (B) Measurement by ELISA assay of the quantity of CCL7secreted by perigonadal adipose tissue explants from normal-weight orobese C57BL/6 mice (high-fat diet). Migration of PC-3 cells againstconditioned medium from adipose tissue explants from normal-weight orobese mice optionally in the presence of CCR1/CCR3 inhibitor (200 nMUCB35625) or 10 μg/ml of control IgG1, anti-CCR1 or anti-CCR3 antibody.(C) Measurement by ELISA assay of the quantity of CCL7 secreted byprimary adipocytes or by Stromal Vascular Fraction (SVF) cells isolatedfrom perigonadal adipose tissue from normal-weight or obese C57BL/6 mice(high-fat diet). Migration of PC-3 cells against conditioned medium fromprimary adipocytes or SVF cells isolated from adipose tissues from obeseor normal-weight mice. Migration of PC-3 cells against CM-Ad fromisolated primary adipocytes optionally in the presence of CCR1/CCR3inhibitor (200 nM UCB35625) or 10 μg/ml of control IgG1, anti-CCR1 oranti-CCR3 antibody.

FIG. 4: Role of the receptor CCR3 in the migration of TRAMP-C1P3 cells.(A) Expression of the receptors CCR1 and CCR3 by TRAMP-C1P3 cells byflow cytometry. Migration of TRAMp-C1P3 cells against medium(serum-free) optionally supplemented with the human recombinantchemokine CCL7 (1-100 ng/ml). Migration of TRAMP-C1P3 cells againstCM-Ad (3T3-F442A) after treatment with CCR1 and CCR3 inhibitor (200 nMUCB35625) and with 10 μg/ml of control IgG1, anti-CCR1 or anti-CCR3antibody. (B) Expression of the receptor CCR3 by TRAMP-C1P3 cellsnon-transfected (WT) or transfected with a control plasmid (sh Ctrl),with 3 independent shRNA targeting the receptor CCR3 (m4, m5 and m6CCR3)by flow cytometry (with quantification of the mean fluorescenceintensity). (C) Migration of transfected TRAMP-C1P3 cells (shCtrl, shRNAm4CCR3, shRNA m6CCR3) against CM-Ad (3T3-F442A) optionally in thepresence of UCB35625 (200 nM). Migration of transfected TRAMP-C1P3 cells(shCtrl, shRNA m6CCR3) against conditioned medium of adipose tissue ofobese or normal-weight mice optionally in the presence of 10 μg/ml ofcontrol IgG1, anti-CCR1 or anti-CCR3 antibody.

FIG. 5: Role of the receptor CCR3 in tumor progression in vivo. (A)Measurement of the tumoral growth of TRAMP-C1P3 cells (shCtrl andshCCR3) 20 days after intraprostatic injection of 2 million cells innormal-weight and obese C57BL/6 mice. (B)

Observations of cross-sections of these tumors after labeling withhematoxylin/eosin (H&E) at different magnification factors (10× zoom).

FIG. 6: The expression of the receptor CCR3 is correlated with theaggressiveness of human prostate tumors. Study conducted using twoseries of prostate cancer TMA, a first collection including 92 tumors(A) and a second annotated collection containing 101 tumors (B). Theexpression of CCR3 was measured by immunohistochemistry and arepresentative result for the healthy epithelium and each group oftumors: low-grade (Gleason<7), intermediate-grade (Gleason 3+4 and 4+3)and high-grade (Gleason>7) tumor, is shown (A and B, left-hand panels).In both series, the expression of CCR3 is strongly correlated with theGleason scores which reflects the aggressiveness of the prostate cancer.For each cohort analyzed, a representation in quartile form is featuredalong with a correlation test (Spearman test).

FIG. 7: Role of the CCR3/CCL7 pathway in the migration of prostate tumorcells in response to mouse medullary adipocyte secretions (bonemetastasis context). (A) Development of two medullary adipocyte models:one in vitro model with the 14F1.1 line and one ex vivo model obtainedfrom the differentiation of mouse mesenchymal medullary stem cells.During the treatment with the selection and differentiation medium,these cells change appearance and are charged with lipids untilmedullary adipocytes are obtained after 6 weeks of ex vivodifferentiation. (B) Comparison of the secretion of CCL7 betweenvisceral primary adipocytes of C57B16 mice, 3T3-F442A adipocytesdifferentiated in vitro, 14F1.1 cells differentiated in vitro and mousemedullary adipocytes differentiated ex vivo. Migration of PC-3 cellsagainst conditioned medium from adipocytes obtained from the differentmodels: differentiated F442A, differentiated 14F1.1 and mature mousemedullary adipocytes (ex vivo). Migration of PC-3 against conditionedmedium from mature mouse medullary adipocytes (ex vivo) optionally inthe presence of CCR1/CCR3 inhibitor (200 nM UCB35625) or 10 μg/ml ofcontrol IgG1, anti-CCR1 or anti-CCR3 antibody. (C) Expression of thechemokine receptors CCR1 and CCR3 by flow cytometry in RM1-BM cells.Migration of RM1-BM against CM-Ad from F442A optionally in the presenceof CCR1/CCR3 inhibitor (200 nM UCB35625) or 10 μg/ml of control IgG1,anti-CCR1 or anti-CCR3 antibody. Migration of RM1-BM cells againstserum-free medium optionally supplemented with the recombinant chemokineCCL7 (100 μg/ml). Migration of RM1-BM against conditioned medium frommouse medullary adipocytes differentiated ex vivo (ex vivo) optionallyin the presence of CCR1/CCR3 inhibitor (200 nM UCB35625) or 10 μg/ml ofcontrol IgG1, anti-CCR1 or anti-CCR3 antibody.

FIG. 8: The expression of CCR3 is higher in tumors of patientspresenting with surgical treatment failure regardless of the Gleasonscore. (A) Scatchard diagram comparing the expression values of CCR3 intumors from patients presenting with surgical treatment failure or not.We used a Student's test to correlate the expression of CCR3 with theoccurrence or not of surgical treatment failure (which is a non-ordinalvariable representing a category). The median values of the expressionof CCR3 for both categories are demonstrated. (B) Scatchard diagramcomparing the expression values of CCR3, after one year of follow-up,with the 4 Gleason score classes. The CCR3 values are systematicallyhigher in patients presenting with surgical treatment failure regardlessof the Gleason score class. The deviation tends to be greater for tumorshaving a low Gleason score (<7).

FIG. 9: Human medullary adipocytes have the ability to chemoattracthuman prostate tumor cells, this effect is dependent on the CCR3/CCL7pathway and is regulated by obesity. (A) Migration of PC-3 humanprostate tumor cells against serum-free medium, containing 10% serum, orconditioned medium from human medullary adipocytes obtained from yellowmarrow samples (CM-MAd). (B) Analysis of the secretion of CCL7 inconditioned media from subcutaneous, periprostatic and human medullaryadipose tissues by ELISA (1 g of tissue in 8 ml of medium for 24 hr).(C) Migration of PC-3 cells against conditioned medium of humanmedullary adipocytes obtained from yellow marrow samples (CM-MAd) in thepresence of CCR1/CCR3 inhibitor (200 nM UCB35625). (D) Migration of PC-3cells against conditioned medium from human medullary adipocytesobtained from yellow marrow samples in the presence of IgG1 and blockingantibodies targeted against CCR3 and CCL7. (E) Analysis of the secretionof CCL7 in conditioned media of human subcutaneous and medullary adiposetissues obtained from non-obese (BMI<25 mg/m²) or obese (BMI>30 mg/m²)by ELISA (1 g of tissue in 8 ml of medium for 24 hours). (F) Migrationof PC-3 cells against conditioned medium of human medullar adipocytesobtained from yellow marrow samples (CM-MAd) from obese or normal-weightpatients in the presence of CCR1/CCR3 inhibitor (200 nM UCB35625).

EXAMPLE Inhibition of Chemokine Receptor CCR3 in Prostate CancerDissemination Materials and Methods Antibodies, Inhibitors and ProductUsed

The monoclonal antibodies targeted against CCR1 (D063-3 clone 141-2) andCCR3 (D083-3, clone 444-11) are obtained from MBL International(Worburn, Mass., USA). The anti-CCR2 (clone E68) and anti-CCR3 (cloneY31) monoclonal antibodies are obtained from Abcam (Cambridge, Mass.,USA). The monoclonal antibodies against CXCR2 (clone 48311), CXCR4(clone 44716) and CCR1 (clone 53504) are obtained from R&D Systems(Minneapolis, Minn., USA).

The inhibitor targeting the receptor CXCR4, AMD3100 (Rosenkilde, Gerlachet al. 2004) is obtained from Sigman Aldrich and is used at finalconcentrations of 1, 10, 50 and 100 nM. The inhibitor targeting thereceptors CXCR1 and CXCR2, SB225002 (White, Lee et al. 1998)) isobtained from Tocris (Bristol, United Kingdom) and is used at finalconcentrations of 1, 10 and 100 nm. The inhibitor targeting the receptorCCR2, sc-202525 (Cherney, Mo et al. 2008) is obtained from Santa CruzBiotechnology (Dallas, Tex., USA) and is used at final concentrations of1, 10 and 50 and 100 nm. The inhibitor targeting the receptor CCR1 andCCR3, UCB35625 (Sabroe, Peck et al. 2000) is obtained from Tocris(Bristol, United Kingdom) and is used at final concentrations of 1, 50,100 and 200 nM.

Cell Culture

The prostate cell lines LNCaP (ATCC® CRL-1740™), DU-145 (ATCC® HTB-81™),PC-3 (ATCC® CRL-1435™), and the human carcinoma cell lines from thecolon sw480 (ATCC® CCL-228™) and sw620 (ATCC® CCL-227™), pancreas CAPAN(ATCC® HTB-79™) and PANC-I (ATCC® CRL-2547™), breast ZR-75-1 (ATCC®CRL-1500™) and MDA-MB321 (ATCC® CRM-HTB-26™) and melanomas SK-Mel-28(ATCC® HTB-72™) and Lu1205 (donated by Dr. Herlyn, Wistar Institute,Philadelphia, USA) are cultured in RPMI culture medium (Invitrogen,Auckland, NZ) supplemented with 10% FCS (Fetal Calf Serum), 2 mML-glutamine, 125 μg/ml of streptomycin, 125 U/ml of penicillin. All thelines used in this study are cultured in a wet atmosphere with 5% CO₂.

The mouse tumor line TRAMP-C1P3 derived from the line TRAMP-C1 (ATCC®CRL-2730™) is cultured in DMEM medium supplemented with 10% FCS, 125μg/ml of streptomycin, 125 U/ml of penicillin, 5 mg/ml of insulin and 10nM of dihydrotestosterone (18).

The RM1-BM line is cultured in DMEM medium (Invitrogen, Auckland, NZ)containing 4.5 μg/l of glucose, supplemented with 10% FCS, 125 μg/ml ofstreptomycin and 125 U/ml of penicillin. The RM1-BM line was supplied byDr. Carl Power (Eastern Virginia Medical School). It consists of asyngeneic mouse cell line having a C57BL/6 genetic background andderived from the RM1 line and which is characterized by the high bonemetastatic potential thereof. Indeed, following an intracardialinjection to C57BL/6 mice, bone metastases are thus obtained in 86% ofcases (Kanno, Amakasu et al. 1994).

The mature adipocytes are obtained from the in vitro differentiation ofthe mouse pre-adipocyte line 3T3-F442A (00070654 Sigma Aldrich)(Kuri-Harcuch and Green 1978). These cells are cultured in DMEM culturemedium (Invitrogen, Auckland, NZ) containing 10% FCS, 125 μg/ml ofstreptomycin and 125 U/ml of penicillin (complete DMEM medium). Toinduce adipocyte differentiation, 6×10⁴ cells are inoculated in a 6-wellplate. After 3 days of culture, the confluent cells are incubated in thepresence of complete DMEM medium containing 50 mM of insulin (referredto as adipocyte differentiation medium) for a period of 10 to 14 days.After 10 days of culture under these conditions, 80% of the cellsexhibit lipid droplets specific for adipocyte differentiation.Conditioned medium from adipocytes (CM-Ad) (1% BSA, 0% FCS) is obtainedduring the culture of the mouse adipocytes differentiated in vitro underthe conditions described above.

During this study, an in vitro medullary adipocyte model was used: theline 14F1.1. This line is derived from immortalized bone marrow cellsselected for the ability thereof to differentiate either intoendothelial cells, or into medullary adipocytes (Yamamura, Salomon etal. 2011) and (Menzies and Salomon 2011). These cells are cultured inDMEM culture medium (Invitrogen, Auckland, NZ) containing 10% FCS, 125μg/ml of streptomycin and 125 U/ml of penicillin (complete DMEM medium).Adipocyte differentiation is obtained 28 days after the cells havereached confluence.

Ex Vivo Differentiated Medullary Adipocyte Model

In order to obtain medullary adipocytes ex vivo, a protocol wasdeveloped for the differentiation of the stromal vascular fraction bonemarrow cells into medullary adipocytes in C57BL/6 mice. In order toisolate these cells, the tibias and femurs of C57BL/6 mice are isolatedand then placed in DMEM (1% BSA) at 4° C. The bones are then cut at bothends and, using a syringe, the cells contained in the bone marrow areretrieved by injecting cold medium inside the bone. The cellularsolution obtained is treated with a 2.5 μg/ml solution at a rate of 5 μLper 2 mL of cellular suspension. The mixture is left to incubate in awater-bath at 37° C. for 5 minutes. This step makes it possible toremove any traces of extracellular matrix. The liberase is thenneutralized with a volume-by-volume addition of DMEM (10% FCS), and thecellular solution is filtered to remove debris and blood clots. Thecellular suspension is centrifuged at 2000 RPM for 20 minutes and thesediment containing the stromal fraction cells is resuspended in DMEMmedium containing 10% FCS, 125 μg/ml of streptomycin and 125 U/ml ofpenicillin. The cells are inoculated in 24-well plates at a density of1-2×10⁷ cells/mL and placed in culture at 37° C. with 5% of CO₂. Thecells are left in culture in this way for 4 days and the culture mediumis then changed so as only to retain the cells having adhered. Once thecells have reached confluence, the medium is replaced by adifferentiation medium. This medium is composed of DMEM (10% FCS)supplemented with 1 pM dexamethasone, 0.45 mM IBMX, 60 pM indomethacinand 10 μg/ml insulin. The medium is changed every 4 days for 30 to 35days. After this period, the mesenchymal precursors have differentiatedinto medullary adipocytes and contain lipid droplets visible undermicroscopy. Conditioned medium from ex vivo medullary adipocytes (CM-Admed) (1% BSA, 0% FCS) is obtained as described above.

Human Medullary Adipocyte Samples

The samples of human yellow bone marrow and human subcutaneous adiposetissue were collected during the implantation of hip or knee prosthesescarried out in the

Traumatology department at Hopital Pierre Paul Riquet (Toulouseuniversity hospital, France). Samples were collected in line with therecommendations of the Midi-Pyrenees (France) ethics and patientprotection committee. Non-opposition to sample collection was obtainedfrom each patient, prior to the operation, in accordance with article L.1211-2 of the French Public Health Code. These adipose tissues areimmediately placed at 37° C. in DMEM (serum-free) medium supplementedwith 1% Bovine Serum Albumin, at a rate of 1 g of tissue per 8 mL ofmedium. The tissues are left to secrete for 24 hours and the mediumcontaining the secretions is retrieved and frozen.

Human Visceral Adipose Tissue Samples for Analysis of the Expression ofCCL7 by Q-PCR

The human visceral adipose tissues (AT-Visc) were collected according tothe guidelines of the ethics committees of the Toulouse-Rangueil andNancy-J. d'Arc hospitals (France). All the patients gave their informedconsent to take part in the study and the research was conducted inaccordance with the Declaration of Helsinki revised in 2000(http;//www.wma.net/e/policy/b3.htm). The human samples of AT-Visc(intra-abdominal) from normal-weight subjects were obtained from 8patients (aged 40.3±2.2 years and body mass index (BMI) of 23.3±1.2kg/m2) having undergone an intra-abdominal surgical procedure. Thesamples of AT-Visc from obese subjects were obtained from 21 patientssuffering from grade III morbid obesity (aged from 41.3±2.2 years andBMI of 45.7±2.9 kg/m2) prior to bariatric surgery. The tissue sampleswere immediately frozen in liquid nitrogen after sampling and stored at−80° C.

Human Periprostatic Adipose Tissue Samples

The periprostatic adipose tissue samples were collected from radicalprostate resection in accordance with the recommendations of the ethicscommittee of Rangueil university hospital (Toulouse, France). All thepatients gave their informed consent to take part in this study whichwas conducted in accordance with the Declaration of Helsinki revised in2000. The periprostatic adipose tissue samples are sampled in thevicinity of the tumor and do not exhibit visible fibrosis. This adiposetissue is immediately placed at 37° C. in DMEM medium (serum-free)supplemented with 1% Bovine Serum Albumin.

Human Yellow Marrow and Subcutaneous Adipose Tissue Samples

The samples of human yellow bone marrow and human subcutaneous adiposetissue were collected during the implantation of hip or knee prosthesescarried out in the Traumatology department at Hopital Pierre Paul Riquet(Toulouse university hospital, France). Samples were collected in linewith the recommendations of the Midi-Pyrenees (France) ethics andpatient protection committee. Non-opposition to sample collection wasobtained from each patient, prior to the operation, in accordance witharticle L. 1211-2 of the French Public Health Code. These adiposetissues are immediately placed at 37° C. in DMEM (serum-free) mediumsupplemented with 1% Bovine Serum Albumin, at a rate of 1 g of tissueper 8 mL of medium. The tissues are left to secrete for 24 hours and themedium containing the secretions is retrieved and frozen.

Mouse Perigonadal Adipose Tissue Samples

Male C57BL/6 mice supplied by Janvier (Le Genest St-Isle, France) aged 8weeks and accommodated in ventilated Plexiglas cages (3 mice per cage)were used. These C57BL/6 mice are either placed on a normal dietconsisting of 5% fat (PicoLab Rodent Diet 20; Purina Mills Inc.,Brentwood, Mo., USA), or, on a high-fat diet (60% fat, 22% carbohydratesand 18% protein) for 10 weeks. All the procedures were validated by theMidi-Pyrenees (France) animal ethics committee. At the end of the diet,the animals are sacrificed by cervical dislocation, weighed and theperigonadal adipose tissue (AT-PG) is sampled and immediately placed at37° C. in DMEM (serum-free) medium supplemented with 1% Bovine SerumAlbumin.

Measurement of Genic Expression by Q-PCR

The total RNA are extracted using the RNeasy® Mini kit (Qiagen GmbH,Hilden, Germany). A total RNA microgram is transcribed to cDNA bySuperscript II reverse transcriptase (Invitrogen, Auckland, NZ).Incubation is conducted in parallel under the same conditions for eachsample in the absence of

Superscript II to ensure that there is no contamination with the genomicDNA. The real-time PCR reaction is performed with 50 ng of cDNA, 5 μl ofan oligonucleotide solution targeting the mouse gene of CCL-7 and 10 μlof SYBR Green TaqMan Universal PCR master mix (Applied Biosystems,Foster City, Calif.). The PCR reaction is first conducted on the genesof mixtures (GAPDH and HPRT) in order to normalize the genic expression(use of GeNorm software). Analyses then related to the expression ofCCL7 gene (human or mouse). The nucleotide sequences of the primers andthe optimal concentrations for use are compiled in table 1 below.

TABLE 1 primers used for Q-PCR Gene Sense primer Antisense primerConcentration studied Species (SEQ ID NO:) (SEQ ID NO:) used CCL7 HumanAAACCTCCAATTCTCA CAGAAGTGCTGCAG 900 Nm TGTGGAA (4) AGGCTT (5) CCL7 MouseAAGATCCCCAAGAGG CTTCCCAGGGACAC 900 Nm AATCTCAA (6) CGACTA (7) GADPHHuman/ TGCACCACCAACTGCT GGCATGGACTGTGG 500 Nm Mouse TAGC (8) TCATGAG (9)HPRT Human/ TGACACTGGCAAAAC GCTTGCGACCTTGA 900 Nm Mouse AATGCA (10)CCATCT (11) HPRT Mouse TGGCCATCTGCCTAGT GGACGCAGCAACT 300 nM AAAGC (12)GACATTTC (13)Digestion of Adipose Tissue with Collagenase

The human or mouse adipose tissue is digested with liberase (RocheApplied Science, Meylan, France) for approximately 30 minutes at 37° C.under stirring. After digestion, liberase is inhibited by adding serumin the medium containing the cells. Adipocytes, due to the lipid contentthereof, float whereas stromal vascular fraction (SVF) cells sink andare found at the bottom of the tube. The isolated primary adipocytes andthe SVF cells are then cultured in DMEM (serum-free) medium containing1% BSA for 24 hours. Conditioned medium is sampled after 24 hours ofsecretion. The cells are counted at the end of the experiment to referthe various secretions to the number of cells.

Human and Mouse CCL7 Assay Using ELISA Test

The human and mouse CCL7 assays were conducted using the ELISA kitsupplied by Peprotech and R&D system respectively in accordance with theprotocols provided by these manufacturers.

Analysis of Expression of Chemokine Receptors in Tumor Lines by FlowCytometry

The tumor cells (from prostate, breast, colon, pancreas, or melanoma)were washed three times in FACS buffer (PBS supplemented with 0.5% BSAand 2% FCS) and fixed with a 3.7% paraformaldehyde solution for 20 minat 4° C. The tumor cells were suspended with anti-CXCR4 (10 μg/ml),anti-CXCR1 (10 μg/ml), anti-CXCR2 (1:25), anti-CCR1 (10 μg/ml),anti-CCR2 (20 μg/ml), anti-CCR3 (10 μg/ml) antibodies or isotypicalmouse anti-IgG control antibodies at 4° C. for 2 hours. The tumor cellswere incubated with a mouse anti-IgG secondary antibody (dilution to1:200) coupled with Alexa Fluor 488 or coupled with Cyanine 5 (for cellsexpressing GFP) for 30 minutes at 4° C. Finally, 2×10⁴ cells wereanalyzed by flow cytometry using a FACScan and FACScalibur flowcytometer (analysis on CellQuest™ software (BD-PharMingen)).

Boyden Chamber Migration Tests

In order to evaluate the ability of tumor cells to migrate according tothe culture medium, Boyden chamber migration tests were conducted using8 μm porosity Greiner bio-one inserts. In the lower compartment eitherculture medium with or without serum, or conditioned medium from F442Aadipocytes (CM-Ad), human periprostatic adipose tissue (CM-PPAT), mouseperigonadal adipose tissue (CM-PGAT), adipocytes or vascular fractioncells (SVF) isolated from PPAT or PGAT is introduced. These conditionedmedia may be pretreated for 30 minutes by adding antibodies blocking thechemokine CCL7. Experiments with the recombinant chemokine CCL7 werealso conducted, in this case the chemokine is added to serum-freeculture medium to verify the chemoattractant potential thereof on a cellline. In the upper chamber 1×10⁵ cells, previously depleted for 6 hoursof serum are deposited in serum-free culture medium. For the migrationtests in the presence of blocking antibodies specific for CCR1 or CCR3or inhibitors of CCR1/CCR3, CCR2, CXCR1/CXCR2 and CXCR4, the tumor cellsare previously incubated in a tube with the blocking antibody or theinhibitor for 30 min at 37° C. The migration of the tumor cells isevaluated after 12 hrs of migration at 37° C. and 5% CO₂ by 1% toluidineblue staining supplemented with 0.1M borax (Sigma, St Louis, Mo., USA)and quantification of the absorbance (measured at 570 nm).

Adipocyte Secretome Analysis (Mass Spectrometry)

The conditioned media from adipocytes (CM-Ad) are sampled after 24 hoursof secretion (medium free from serum and phenol red) centrifuged andfiltered to remove the cell debris and supplemented with proteaseinhibitor. The concentration of 5 ml of adipose CM-Ad medium wasconducted with StrataClean resin (Agilent Technologies, Santa Clara,Calif., USA) according to the manufacturer's instructions.

After reduction and alkylation of cysteine, the sample was separated bySDS-PAGE (12% acrylamide). The proteins were visualized by Coomassieblue staining and each track was divided into 13 homogeneous bands anddigested (digestion of gel by trypsin). The digestion product wasanalyzed by nanoLC-MS/MS via the Ultimate3000 system (Dionex, Amsterdam,Netherlands) coupled with an LTQ-Orbitrap mass spectrometer (ThermoFisher Scientific, Bremen, Germany) as described above (Contreras,Ferrero Guadagnoli et al. 2010), except that the peptides were elutedusing a 5 to 50% gradient of solvent B for 60 min at a flow rate of 300nL/min. Mascot was used to automatically extract the “peak lists” fromthe raw files. The MS/MS data were compared to all the entries in themouse databases (Mus musculus) and the identification of the peptideswas validated using a computer system as described above (Santini,Salomon et al. 2010), except that 1% FDR was used for the validationpeptides and proteins.

Inhibition of the Expression of CCR3 Using shRNA

In order to inhibit the expression of the receptor CCR3 (NM 009914) in astable manner in TRAMP-C1P3 cells, an shRNA approach was used enablingstable negative regulation even after several months of culture. Theinterfering sequences were generated using the Dharmacon “CentersiDesign” tool. The sequences of the sense strands of shRNA generatedare as follows:

m4CCR3 (SEQ ID NO: 1): 5′-cgcgtccccAGACCACACCCTATGAATAttcaagagaTATTCATAGGGTGTGGTCTtttttggaaat-3, m5CCR3 (SEQ ID NO: 2):5′-cgcgtccccGACCACACCCTATGAATATttcaagagaATATTCATAGGGTGTGGTCtttttggaaat-3 and m6CCR3 (SEQ ID NO: 3)5′-cgccgtccccGGTGAGAGGTTCCGGAAAttcaagagaGTTTCCGGAACCTCTCACCtttttggaaat-3.

The nucleotides targeting the sequence of CCR3 are shown in upper case,whereas the sequence responsible for the hairpin structure and thesequences required for directional cloning are represented in lower caseletter. The lentiviral vector LVTHM® is a lentiviral vector (LV) codingfor the shRNA CCR3 sequences under the control of the H1 promoter alsoexpressing GFP. The TRAMP-C1P3 cells were then transfected in thepresence of m4, m5 or m6-LVTHM for 24 hours at a multiplicity ofinfection (MOI) of 1:10. The transfected population is selected by GFPexpression (cell sorting). These cells were then analyzed by flowcytometry to validate the loss of expression of the CCR3 gene. Thecontrol used is the μLVTHM plasmid which expresses an ineffective shRNAsequence.

Intraprostatic Injection of TRAMP-C1P3 Cells in C57BL/6 Mice

Male C57BL/6 mice aged 18 weeks on a normal or high-fat diet wereanesthetized with isoflurane. A transverse incision was made at thelower part of the abdomen. The muscles of the abdominal wall wereincised, and the bladder and the seminal vesicles were released so as toexpose the dorsal lobe of the prostate. A suspension of 2×10⁶ cells (in30 μl of PBS) was then carefully injected under the prostate capsule bymeans of a gauge 30 needle. The incision was closed using 4-0 suture andclips. The animals were then placed in cages and regular monitoring isconducted in order to monitor macroscopically the general condition ofthe mouse. After 20 days of tumor establishment, the mice aresacrificed, the primary tumor is thus removed, measured and weighed, andan acquisition in white light and GFP is carried out. The tumors arethen placed in a paraformaldehyde solution (4%) and included inparaffin.

Sampling of Human Prostate Tumor Samples and Tissue Micro-Arrays TMA

The first database includes a cohort of 92 patients all operated on forprostate adenocarcinoma in the Urology Department of Toulouse universityhospital (France).

For the second database, it consists of a single-center, prospectivestudy. The patients included were all operated on for prostateadenocarcinoma, between Feb. 1, 2010 and Dec. 1, 2011, in the UrologyDepartment of Toulouse university hospital (France). The surgicaltreatment consisted of a radical prostate resection by therobot-assisted laparascopic route or by the open retropubic route. Theprostate resection was associated in some cases with bilateralilio-obuturator lymph node dissection (standard or extended). All thepatients included in the study presented with localized disease with nometastatic site during the procedure. For all the patients, a form inrespect of non-opposition to the use of their tissue samples forscientific purposes was collected and signed prior to the procedure inorder to be included in a study. The fresh specimen, accompanied by thecertificate of non-opposition, is processed within 15 minutes followingthe surgical exeresis in order to obtain the shortest possible intervalbetween devascularization and freezing to ensure preservation of thelabile molecules. The tissue samples collected are placed in cryotubesand frozen in nitrogen and stored at −80° C.

In order to facilitate the study, a multiplex histological analysis waspossible due to the performance of “tissue microarrays” (TMA) on regionsof interest selected on prostate sample slides. The regions of interestretained for TMA preparation were selected by two pathologists indouble-blind mode: the aim was to select a region rich in carcinomatoustissue. The tumor regions selected corresponded morphologically tosectors having a comparable histological differentiation to the Gleasonscore of the tumor or to the least favorable Gleason score in the caseof a multifocal tumor. After having chosen the blocks to be sampled,core samples of 0.2 mm in diameter were included on an orthonormal basisalong a predefined plane in a receiving block. The receiving block couldthen be cut on a standard microtome.

Analysis of the Expression of CCR3 by Tissue Immunohistochemistry

Immunohistochemistry experiments on human prostate tumor biopsies wereconducted in order to detect the expression of

CCR3. Immunolabeling was conducted using EnVision™ FLEX Mini Kit, HighpH (Dako Autostainer/Autostainer Plus) (Dako France, Trappes, FR). TheTMA (Tissue Micro-Arrays) are immersed in xylene to remove the paraffinand then rehydrated with successive graduated alcohol baths (100 to 70%followed by distilled water), followed by a treatment with antigenunmasking solution for antigen retrieval (Dako1× pH 6 citrate bufferwith 95° C. water-bath). After an endogenous peroxidase saturation step(Peroxidase blocking, Dako), the samples are incubated with theanti-CCR3 primary antibody (diluted to 1:100). The TMA are thenincubated with the biotinylated secondary antibody, then withHRP-conjugated streptavidin (Dako). They are then rinsed, and treatedwith liquid DAB (BioGenex, San Ramon, Calif.), and washed with distilledwater. Finally, counterstaining was performed with hematoxylin (Dako),and the various TMA are finally mounted using EUKITT reagent. The slidesare digitally scanned using the Hamamatsu Nanozoomer 2.0RS apparatus andanalyzed using the software supplied by the manufacturer. The TMAreadings were conducted by an anatomopathologist.

Measurement of Surface Area of Periprostatic Adipose Deposits

The measurement of the surface area of the periprostatic adiposedeposits was conducted on 15 patients from the main cohort used in ourstudy. The measurements were conducted using preoperative CT-scan or MRIimaging data of each of these patients. For this, images ofcross-sections at the femur were used, at the point where the fusion ofthe symphysis pubis starts. The adipose deposit which was studied issituated between the anterior part of the prostate and the symphysispubis. It is easily recognizable by the difference in attenuationbetween the bone, the prostate and the adipose tissue. The thickness andwidth of the deposit were measured using these images. The area of thisdeposit was then approximated for each patient, by calculating anelliptical area based on the data collected.

Statistical Analysis

The statistical significance of the differences between the means (atleast three independent experiments) was evaluated using Student tests,performed using Prism software (GraphPad Inc.). For the analyses on thepatient, Spearman rank correlation tests were conducted. Thecorrelations are described as strong for values greater than 0.7,moderate for values between 0.3 and 0.7 and weak for values less than0.3. The values of p less than 0.05 (*), <0.01 (**) and <0.001 (***) areconsidered to be significant whereas “NS” signifies non-significant.

Results

The Receptor CCR3 is Involved in the Migration of Prostate Tumor CellsAgainst Conditioned Medium from Adipocytes

Boyden chamber migration tests were performed with prostate cancer linesof increasing aggressiveness (LNCaP, C4-2B, Du-145 and PC-3). The lowerchamber contains different media: RPMI free from fetal calf serum(negative control), RPMI containing 10% fetal calf serum (positivecontrol), conditioned medium from mature adipocytes (CM-Ad) (obtainedfrom the in vitro differentiation of the mouse pre-adipocyte line3T3-F442A (Kuri-Harcuch and Green 1978). The percentage of migrant cellsagainst a medium containing 10% serum is dependent on tumoralaggressiveness. The percentage of migration in the presence of CM-Ad issimilar to the migration induced by medium containing 10% serum for theLNCaP and C4-2B lines and is greater for the Du-145 and PC-3 lines(p<0.05), demonstrating that this medium contains chemokines (see FIG.1A).

As described above (Vindrieux, Escobar et al. 2009), the receptors CCR2,CXCR1, CXCR2 and CXCR4 are expressed by the prostate cancer lines Du-145and PC-3, the expression detected by flow cytometry. The receptor CCR3is expressed in the prostate cancer lines Du-145 and PC3 unlike thereceptors CCR1 (see FIG. 1B).

The functionality of these receptors in the migration of PC-3 cellsagainst CM-Ad was tested using known specific inhibitors used at theinhibitory doses described in the literature. The results (see FIG. 1C)are as follows:

AMD3100 (specific inhibitor of CXCR4; Rosenkilde et al., 2004)significantly reduces by approximately 20% (p<0.05) the migration ofPC-3 cells against CM-Ad medium only at the maximum dose of 100 nM;

SB225002 (inhibitor of CXCR2 and CXCR1 at a lower affinity; White etal., 1998) significantly inhibits by approximately 30% the migrationinduced by CM-Ad at the dose of 50 nM (p<0.01);

sc-202525 (specific antagonist of CCR2; Cherney et al., 2008) inhibitsby merely 10% the migration of tumor cells (p<0.05);

UCB35625 (inhibitor of CCR1 and CCR3; Sabroe et al., 2000) induces asignificant dose-dependent decrease in the migration of PC-3 tumor cellsinduced by CM-Ad from 50 nM (p<0.05). The maximum effect is observed at200 nM (approximately 50% inhibition, p<0.001). These results wereconfirmed by a more specific approach based on the use of antibodiesblocking CCR1 (reference D063-3, clone 141-2, MBL international Corp,(Iacono, Masciangelo et al. 1994)) or CCR3 (D083-3, clone 444-11, MBLinternational, (Iacono, Masciangelo et al. 1994)). The incubation of thetumor cells with a blocking antibody targeted against CCR3 (10 μg/ml)induces a significant inhibition of the migration induced by CM-Ad(approximately 50%, p<0.01) with respect to the isotypical control. Onthe other hand, no inhibition effect is observed with the use ofblocking antibody targeted against CCR1, which is not expressed by thetumor cells.

The specificity of the role of CCR3 in the migration of prostate cellsagainst conditioned medium from mature adipocytes was studied. Theresults are shown in FIG. 1D. Various epithelial tumors were used, forwhich adipose tissue (AT) is situated anatomically in the vicinity whenthey become invasive. It consists of breast cancer (mammary AT),pancreatic and colon cancer (visceral AT). Furthermore, melanoma lineswere included; this tumor when invading finding itself in contact withthe AT of the hypodermis. Two pairs of lines were chosen for each tumortype. As shown in FIG. 1D (left-hand panel), breast (T47D andMDA-MB231), colon (sw480 and sw620), pancreatic (CAPAN and PANC-1)cancer and melanoma (501Mel and SK28) lines express the receptor CCR3(flow cytometry). All these lines migrate against CM-Ad, the percentageof migrant cells being correlated with the degree of aggressivenessthereof. The migration of pancreatic, colon cancer and melanoma tumorcells against CM-Ad medium is not affected by the presence of UCB35625at a dose of 200 nM (right-hand panel). Only a very modest effect(inhibition of the order of 10%, p<0.05) is observed for the aggressivebreast cancer line MDA-MB231, whereas the migration of the breast cancerline (T47D) is not affected. In this same series of experiments,significant inhibition of migration against CM-Ad medium of PC-3- andDu-145 prostate tumor cells by approximately 50% is detected (p<0.001).

The Ligand of CCR3, CCL7 is Involved in the Migration of Prostate TumorCells Against Conditioned Medium of Adipocytes

A proteomic analysis was conducted on CM-Ad (Proteomics Department,IPBS, CNRS UMR5089, Toulouse France). This analysis was conducted on 3independent media. Six chemokines were detected reproducibly: CXCL1,CXCL5, CXCL12, CCL2, CCL7 and CCL9 (right-hand panel). The chemokines,CXCL1 and CXCL5, can interact with the receptor CXCR2, CXCL12 with CXCR4and CCL2 with CCR2 (left-hand panel). These results (see FIG. 2A) couldexplain why the inhibitors of CXCR1/CXCR2, CXCR4 and CCR2 partiallyreduce the chemotaxis induced by CM-Ad (FIG. 1C). Of the ligands ofCCR3, only CCL7 is detected. It should also be noted that no ligand ofCXCR1 was detected in the adipocyte secretions analyzed.

The recombinant chemokine CCL7 is capable of inducing the migration ofPC-3 and Du-145 prostate cells in a dose-dependent manner in a Boydenchamber migration test. The results (see FIG. 2B) are significant forboth lines from 50 ng/mL compared to the medium alone (left-hand panel).In these two same cell lines, migration of the prostate cells isinhibited by approximately 50% by a blocking antibody targeted againstCCR3 and by a blocking antibody targeted against CCL7 (AF-456-NA, R&DSystem) (p<0.001), whereas the antibody blocking CCR1 has no effect(right-hand panel).

Perigonadal adipose tissue (AT-mu-PG) was sampled from male mice (3independent experiments conducted on 3 independent lots of mice,containing 3 to 10 mice). Similar experiments were conducted withperiprostatic adipose tissue in humans (AT-hu-PP). This AT is obtainedon prostate resection specimens, remote from the tumor. This tissueexhibits macroscopic characteristics of AT and does not contain tumorcells. These experiments were conducted on 5 patients having a BMIbetween 20 and 25. For these two types of AT, conditioned medium wasprepared using a 24-hour culture. These experiments (see FIG. 2C)demonstrate that CCL7 is secreted by these two AT with a mean of 155±20μg/mL/g of AT in mice and 75±15 μg/ml/g of AT in humans (left-handpanel). The conditioned media of human and mouse AT are capable ofinducing in a similar manner the migration of tumor cells (approximately20% migrant cells for both media). Finally, this migration issignificantly inhibited by the use of inhibitors and antibodies blockingthe CCR3/CCL7 pathway and by the inhibitor of CCR3 (200 nM UCB35625)(right-hand panel). The CCR3/CCL7 pathway is thus involved in themigration of prostate cells against secretions of mouse visceral andhuman periprostatic adipose tissue.

The Secretion of CCL7 by AT is Increased in an Obesity Context whichHelps Amplify the Role of the CCR3/CCL7 Pathway in Prostate CellMigration

The genic expression of CCL7 was evaluated in a series of samples ofhuman (intra-abdominal) visceral AT (VAT) from patients who were eithernormal weight (Body Mass Index (mean BMI 23.3±1.2 kg/m², 8 samples), orobese (mean BMI 45.7±2.9 kg/m², 21 samples). The results (see FIG. 3A)show an approximately 3-fold increase of the mRNA of CCL7 in the VAT ofobese subjects compared to subjects of normal BMI (p<0.01) (left-handpanel). In mice, the level of expression of the mRNA of CCL7 is alsoincreased by a factor of 3 in mice rendered obese by a “High-Fat Diet”(HFD) compared to mice of normal weight (“Normal Diet”, ND) (p<0.001).Similar results were obtained with a mouse genetic obesity modelexhibiting invalidation of the leptin receptor (db/db mice) compared towild mice.

Perigonadal adipose tissue (AT-mu-PG) was sampled from mice subjectedfor 8 weeks, either to a normal diet (normal-weight mice) or a high-fatdiet resulting in the onset of obesity (obese, HFD) (independentexperiments conducted on 3 different lots of mice). The results areshown in FIG. 3B. In the conditioned medium prepared as above, a2.7-fold increase in the level of secretion of CCL7 is observed in thetissue from obese mice compared to normal-weight mice (p<0.05)(left-hand panel). These conditioned media were used to conduct Boydenchamber migration experiments. The migration of PC-3 tumor cells againstconditioned medium is significantly increased by obesity (28% migrantcells against CM of AT of normal-weight mice versus 40% against CM of ATof obese HFD mice, p<0.01) (right-hand panel). Under both conditions,the migration of tumor cells against CM is inhibited by the use ofinhibitors and antibodies blocking the CCR3/CCL7 pathway, the effectbeing however more pronounced under obesity conditions. As such,UCB35625 or anti-CCR3 or CCL7 inhibit migration against CM of NDAT-mu-PG by 25%, whereas this effect is 60% for CM of HFD AT-mu-PG.

AT-mu-PG of ND or HFD mice were digested with collagenase in order to beable to separate, after a short centrifugation, the mature adipocytes(floating cells) from the stromal vascular fraction (containing theendothelial cell, macrophages, adipocyte stem cells and pre-adipocytes,etc.). These mature adipocytes were then incubated for 24 hours in aserum-free medium supplemented with 1% BSA in order to prepare CM. Theassay of CCL7 in these media (3 independent media) was conducted byELISA. The results (see FIG. 3C) demonstrate that the primary adipocytessecrete CCL7 and that this secretion is highly regulated in an obesitycontext (90±10 μg/mL/10⁵ cells versus 720±80 μg/mL/10⁵ cells, p<0.001).The migration of tumor cells against CM of adipocytes from obese animalsis significantly increased with respect to the CM of normal-weightanimals (p<0.01). Under both conditions, the migration of tumor cellsagainst CM is inhibited by the use of inhibitors and antibodies blockingCCR3, the effect being however more pronounced using obesity conditions.As such, the inhibition of CCR3 by UCB35625 or by an antibody reduces by60% the migration against CM of adipocytes from obese mice, whereas thiseffect is 30% for CM of adipocytes from normal-weight mice (p<0.01).

Validation of the Involvement of CCR3 in Migration in Response toAdipocyte Secretions in a Mouse Tumor Line Model (TRAMP-C1P3)

The TRAMP-C1P3 syngeneic mouse line for the mouse strain C57 BL/6 wasused to produce orthotopic prostate tumor cell graft models innormal-weight or obese mice. This choice of line is justified in thatthe C57 BL/6 is a model sensitive to obesity induced by a high-fat dietwith perfectly characterized reproducibility (Winzell and Ahren 2004).The results (see FIG. 4A) demonstrate that TRAMP-C1P3 cells, like humanprostate tumor cells, express the receptor CCR3 but not the receptorCCR1 (left-hand panel). The recombinant chemokine CCL7 is capable ofinducing dose-dependent chemotaxis as for the human lines from 50 ng/mL(center panel). Finally, the migration induced by CM-Ad is significantlyinhibited by the use of UCB35625 (200 nM, p<0.05) of the blockingantibodies targeted against CCR3 (10 μg/mL, p<0.001) or CCL7 (10 μg/mL,p<0.01) whereas the blocking antibody targeted against CCR1 isineffective (right-hand panel).

Three plasmids containing shRNA targeted against the coding sequence ofCCR3 and against a non-coding sequence (control shRNA) were constructed.These plasmids were transfected by a lentiviral approach in theTRAMP-C1P3 line and 3 lines (m4CCR3, m5CCR3, m6CCR3) invalidated for thereceptor CCR3 were obtained along with a control line (shCtrl). Theresults are represented in FIG. 4B. As shown in the left-hand panel(upper quadrant of FIG. 4B), the 3 lines invalidated for CCR3 exhibit asignificant reduction in the expression of the receptor with respect tothe control line (immunofluorescence). This inhibition of the expressionof CCR3 is accompanied by a significant reduction in migration againstCM-Ad for the three shCCR3 lines with respect to the control line(left-hand panel, lower quadrant). This reduction in the migration isalso detected for the m6CCR3 line with respect to the control line whenthe conditioned medium of ND AT-mu-PG is used. Under these conditions,UCB35625 and the antibodies blocking CCR3 or CCL7 no longer have asignificant effect on the migration of m6CCR3 tumor cells unlike thecontrol line (p<0.001). Finally, the increase in migration observed inthe presence of HFD AT-mu-PG is not significant for the m6CCR3 lineunlike the shCtrl line and the effect of UCB35625 and the antibodiesblocking CCR3 or CCL7 is not significant (right-hand panel, lowerquadrant).

The Receptor CCR3 is Involved in Tumor Progression in Mouse Models InVivo

The results are given in FIG. 5A. The injection of the TRAMP-C1P3 lineinto the prostate (dorsal lobe) of 16 week old C57BL/6 mice fed using anormal-fat diet (ND) (mean weight 32±3 g) results in the onset of tumors(measured at 3 weeks, mean tumor size 800±100 mm3, n=14). When the cellsare injected into the prostate gland of mice under an 8-week high-fatdiet (HFD) and which are obese (mean mouse weight: 45±3 g, injection at16 weeks as for normal-weight mice), there is a significant increase inthe tumor size of approximately 2-fold with respect to the non-obesemice (mean tumor size 1500±80 mm3, n=14, p<0.001). The same experimentsconducted with the m6CCR3 line demonstrate a significant decrease in thetumor size with respect to the shCtrl line whether for mice fed with anormal-fat diet (mean weight 30±4 g [NS with respect to the shCtrlgroup], tumor size 220±30 mm3, n=11, p<0.001 [with respect to the shCtrlgroup]) or obese mice fed with a high-fat diet (mean weight 45±3 g) [NSwith respect to the shCtrl group], tumor size 210±25 mm3, n=11, p<0.001[with respect to the shCtrl group]. For mice injected with the m6CCR3line, there is no longer a difference in tumor size between thenormal-weight or obese mice (tumor size 220±30 mm³ versus 200±50 mm³),(left-hand panel in FIG. 5A). An anatomo-pathological analysis of thetumors was conducted (lower panel). The analysis of the histologicalsections of these tumors after hematoxylin-eosin (HE) staining confirmsthat the mice injected with the control line (C1P3 Ctrl) exhibit tumorsof a large volume having completely invaded the dorsal lobe of theprostate. For these tumors, a disappearance of periprostatic adiposetissue is also observed in particular in the obese mice. Indeed, incontact with the tumor, the mature adipocytes are delipidized anddedifferentiated which results in the replacement thereof by a fibroustissue. On the other hand, in the mice injected with the lineinvalidated for CCR3 (C1P3 m6CCR3), tumors of a smaller volume areobserved, characterized by a much less extensive invasion of the dorsallobe of the prostate than for the C1P3 Ctrl line. At the periphery ofthe tumor, intact adipose tissue is found, exhibiting hypertrophy andhyperplasia in the obese mice.

Expression of the Receptor CCR3 is Detected in Aggressive ProstateTumors and the Expression Thereof is Correlated with the Extension ofthe Tumor Outside the Prostatic Capsule

The expression of the receptor CCR3 in human tumors was analyzed in afirst TMA (Tumor Micro-Array) containing 92 prostate tumors. The resultsdemonstrate that CCR3 is expressed in human prostate tumors (figurerepresenting the immunohistochemistry labeling result, left-hand panelin FIG. 6A) and that the level of expression thereof is correlated withthe grade of the tumor defined by the Gleason score (coefficient ofcorrelation of 0.62, p<0.001, Spearman test) (FIG. 6A, right-handcolumn). In low-grade tumors (Gleason score less than 7), a highexpression of CCR3 is observed (quantitative immunohistochemical score+++) in 10% of tumors against 75% of tumors expressing a low CCR3 score(quantitative immunohistochemical score +). On the other hand, in moreaggressive tumors having a Gleason score equal to and/or greater than 7,intense labeling of CCR3 is detected in 46.8% and 76% of tumorsrespectively against 12.8% and 4% exhibiting weak labeling respectively.Finally, it should be noted that the expression of CCR3 is low or absentin non-tumoral prostate glands.

A second series of experiments was conducted on an annotated collectionof 101 patients. The patients included were all operated on for prostateadenocarcinoma in the Urology Department of Rangueil university hospital(France). The TMA were produced in duplicate using a region containingtumor from cryopreserved prostate resection specimens. In order toimprove the quantification of CCR3, the tumor slides were acquireddigitally and the expression of the protein was quantified using ImageJsoftware plugins to obtain more precise measurements than the conventionevaluation based on manual notation (Systel et al., 2013).

Immunohistochemical Quantification of the Vitamin B12 Transport Protein(TCH), Cell Surface Receptor (TCH-R) and Ki-67 in Human TumorXenografts.

The clinical characteristics of the cohorts are given in table 2hereinafter and the immunohistochemical characteristics in table 3hereinafter.

TABLE 2 Clinical characteristics of cohort. Percentage Variables ofcohort Cohort size (number of patients 101 100 Age at time of surgery(years), 63 median (range) (47-75) Body Mass Index (BMI) (kg/m²), 26.1median (range) (19-34) Post-surgery PSA (ng/mL), 7 median (range) (2-37) Latest PSA assay (ng/mL), 0 median (range)    (0-4.02) Prostateresection specimen 48 weight (g), median (range) (22-93) Monitoring time(Day), median 765 (range)   (62-1736) Post-surgery Monitoring 71 70.30treatment only (number of Radiotherapy 16 15.84 patients) only Hormone 76.93 therapy only Radiotherapy 7 6.93 and hormone therapy Post-surgeryBiochemical 14 13.86 progression recurrence (number of Surgical 30 29.70patients) treatment failure ^(a) No recurrence 70 69.31 Death 1 0.99

TABLE 3 Histological and immunohistochemical characteristics of cohort.Percentage Variables of cohort Gleason score, <7 18 17.82 number of =7(3 + 4) 39 38.62 patients +7 (4 + 3) 36 35.64 >7 8 7.92 Percentage ofundifferentiated 40 contingent (grade 4 and 5), (0-100) median (range)Tumor site, Transition 10 9.90 number of zone patients Peripheral 9190.10 zone Tumor stage, pT2b 6 5.94 number of pT2c 35 34.65 patientspT3a 44 42.57 pT3b 16 15.84 Positive surgical margins, 22 21.78 numberof patients No 93 92.08 Presence of Yes 8 7.92 lymphatic emboli, numberof patients Bilateral lymph node 79 79.21 dissection, number of patientsLymph node Nx (No lymph 22 21.78 invasion, node number of dissectionpatients conducted) N0 74 73.27 N1 5 4.95 Expression of CCR3 (arbitrary48.5 units), median (range) (11.4-94.7)

As in the previous cohort, an expression of CCR3 is detected in theprostate tumors (figure representing the immunohistochemistry labelingresult, left-hand panel in FIG. 6A), and the level of expression thereofis correlated with the grade of the tumor defined by the Gleason score(coefficient of correlation of 0.472, p<0.001, Spearman test) (FIG. 6A,right-hand column). An extensive analysis of the correlation between thelevel of expression of CCR3 and various clinical and biologicalparameters of the tumors was conducted (see results hereinafter).

As such, a correlation was demonstrated between the level of expressionof CCR3 and

the Gleason score (p<0.001),

the percentage of poorly histologically differentiated tumor contingent(grade 4 and grade 5 combined) (p<0.01),

the histological pT stage equivalent to the degree of extension of thetumor (p<0.03)

the peripheral site of the tumor (p<0.04),

the present of lymphatic emboli (p<0.02),

biological recurrence defined by two successive PSA assays>0.2 mg/mL(P<0.001).

Furthermore, the level of expression of CCR3 is increased in obesepatients (p=0.01).

The expression of the receptor of CCR3 is a factor of poor prognosis inprostate cancer. There is a significant correlation between the level ofexpression of CCR3 and surgical treatment failure (p<0.001). Surgicaltreatment failure is defined either by biological recurrence (twosuccessive PSA levels greater than 0.2 ng/mL), or by locoregionalrecurrence or the onset of remote metastases, or by the use of hormonedeprivation treatment and/or radiotherapy immediately after surgery, acriterion which was used in a previous study (Malavaud et al., 2010).

Sphingosine Kinase-1 Activity and Expression in Human Prostate CancerResection Specimens.

The CCR3 values were correlated with this criterion (see FIG. 8A). Whenthe CCR3 values were compared in patients presenting with surgicaltreatment failure or not at one year of follow-up in the four Gleasonscore classes (i.e. <7, 7 [3+4 or 4+3], >7), they were systematicallyhigher in patients presenting with surgical treatment failure (FIG. 8B).Furthermore, the differences were greater in the case of a low Gleasonscore (<7). The CCR3 level could thus represent a prognostic factor ofrecurrence.

In one subgroup of patients, the extent of periprostatic adipose tissuewas measured. The clinical (table 4 hereinafter) and histological andimmunohistochemical characteristics (table 5 hereinafter) of thissubgroup are shown.

TABLE 4 Clinical characteristics of cohort for which prostatic fat wasmeasured. Total number of patients (percentage in column; %) 15 (100)Age (Years), Median (range) 62 (48-71) BMI (kg/m²), Median (range) 28(20-34) Initial PSA prior to prostate resection 7 (2-34) (ng/ml), Median(range) Prostate resection specimen weight (g), 48 (32-69) Median(range) Proposed post-surgery Monitoring only, n 10 (66.7) treatment(percentage in column) Radiotherapy, n 3 (20) (percentage in column)Hormone therapy, n 3 (20) (percentage in column) Post-surgery Nobiological 12 (80) progression recurrence Biological recurrence 3 (20)Death 1 (10)

TABLE 5 Histological and immunohistochemical characteristics of cohortfor which prostatic fat was measured. Total number of patients(percentage in 15 (100) column; %) Tumor site Transitional, n 1 (6.7)(percentage in column) Peripheral, n 14 (93.3) (percentage in column)Gleason score on 5, n (percentage in 2 (13.3) prostate resection column)specimen 6, n (percentage in 1 (6.7) column) 7, n (percentage in 10(66.7) column) 8, n (percentage in 1 (6.7) column) 9, n (percentage in 1(6.7) column) Percentage of poorly differentiated 25 (0-95) contingent(grade 4 and 5 combined), Median (range) Tumor volume (prostate invasion13 (6-48) percentage), Median (range) Presence of emboli yes, n(percentage in 2 (13.3) column) no, n (percentage in 13 (56.7) column)pTNM stage pT2a, n (percentage 1 (6.7) in column) pT2b, n (percentage 4(26.7) in column) pT3a, n (percentage 7 (46.7) in column) pT2a, n(percentage 3 (20) in column) pT2a, n (percentage 0 (0) in column)Presence of metastatic lymph nodes **, n 1 (6.7) (percentage in column)Expression of CCR3 0 = none, n 1 (6.7) (percentage in column) 1 = low, n2 (13.3) (percentage in column) 2 = moderate, n 10 (66.67) (percentagein column) 3 = high, n 3 (20) (percentage in column)

Abundant PPAT was defined by a surface area greater than 10 cm2, thesurface area for which all patients exhibit a Gleason score greater than7 (see table 6 hereinafter).

TABLE 6 Correlation between surface area of periprostatic adiposedeposit and various clinical, histological and biochemicalcharacteristics of cohort. Periprostatic adipose tissue surface areaSmall Large Spearman surface surface coefficient area area of p- (<10cm²) (>10 cm²) correlation value Age (years), Median 61 (61-71) 62(48-69) −0.017 0.449 (range) Gleason < 7 3 (50) 0 (0) 0.5843 0.022Gleason Gleason 3 (50) 6 (60) score, n (3 + 4) (in column) Gleason 0 (0)2 (20) (4 + 3) Gleason > 7 0 (0) 2 (20) Non-differentiated 5 (0-25) 25(20-95) 0.461 0.083 contingent, Median (range) Tumor 1 = pT2b 0 (0) 1(10) 0.38 0.163 stage, n 2 = pT2c 2 (40) 2 (20) (in 3 = pT3a 3 (60) 4(40) columns) 4 = pT3b 0 (0) 3 (30) Prostate weight, 52 (44-69) 7.15(6.38-8.96) −0.24 0.39 Median (range) PSA (ng/mL), 6.7 (7-7.5) 7.15(6.38-8.96) −0.163 0.89 Median (range) Biological reascension 1 (20) 2(20) 0.116 0.681 recurrence, n (in column) BMI (kg/m2), Median 28(22-34) 27 (20-31) −0.243 0.3 (range) Overweight and obesity 3 (60) 7(70) −0.224 0.38 (BMI ≥ 25 kg/m2), n (in column) Expression 1 = low, n 2(40) 0 (0) 0.67 0.006 of CCR3 (percentage in column) 2 = 3 (60) 7 (70)moderate, n (percentage in column) 3 = high, n 0 (0) 3 (30) (percentagein column)

The Expression of the Receptor CCR3 Plays a Role in the Migration ofProstate Cancer Cells Induced by Medullary Adipocytes

Two types of models were used: the line 14F1.1 which is a line ofmedullary pre-adipocytes wherein the differentiation (obtained after 21days of confluence) in vitro makes it possible to obtain adipocytescontaining lipids (Zipori, Friedman et al. 1984) (left-hand panel inFIG. 7A). Experiments were also conducted with medullary adipocytesobtained from the in vitro differentiation of progenitors derived fromC57BL/6 mouse bone marrow. These progenitors were cultured for 4 weeksin a differentiation medium containing DMEM (10% serum) supplementedwith 1 pM dexamethasone, 0.45 mM IBMX, 60 pM indomethacin and 10 μg/mlinsulin, making it possible to obtain mature adipocytes containing lipiddroplets (center panel in FIG. 7A).

The CCL7 assay using the ELISA method demonstrates that the conditionedmedia of these cells contain CCL7 at a comparable level to that of3T3F442A differentiated in vitro (3 independent samples). Theseexperiments also made it possible to demonstrate that these medullaryadipocytes secrete quantities of CCL7 greater than those generated by NDvisceral adipocytes (p<0.001). In a Boyden chamber migration tests,medullary adipocytes are capable of inducing the migration of PC-3 tumorcells at a comparable level to that of F442A mature adipocytes (centerpanel). This migration is inhibited by UCB35625 (200 nM) and by the useof antibodies blocking CCR3 and CCL7 (10μg/ml) (p<0.01). The results areshown in FIG. 7B.

The RM1-BM line is a prostate cancer line capable of forming bonesmetastases in 90% of cases in C57BL/6 mice after intracardiac injection(Ohori, Egawa et al. 1994). These cells, similar to all the prostatetumor lines studied, express the receptor CCR3 but not the receptor CCR1as demonstrated by flow cytometry (left-hand panel in FIG. 7C). Therecombinant chemokine CCL7 induces significant migration of RM1-BM cellsat a dose of 100 ng/mL with respect to the control (serum-free medium,p<0.01) (center panel). The migration of RM1-BM cells againstconditioned medium of medullary adipocytes is significantly inhibited byUCB35625 at the dose of 200 nM (p<0.05), and by the blocking antibodiestargeted against CCR3 and CCL7 at the dose of 10 μg/ml (p<0.01)(right-hand panel). The results are given in FIG. 7C.

Human Medullary Adipocytes have the Ability to Chemoattract HumanProstate Tumor Cells, this Effect is Dependent on the CCR3/CCL7 Pathwayand is Regulated by Obesity

The results are given in FIG. 9.

FIG. 9A: From yellow marrow samples from patients obtained inconjunction with the Traumatology department at Hopital Pierre PaulRiquet (Toulouse university hospital, France), the medullary adipocytesare isolated and incubated for 24 hours in serum-free medium containing1% BSA. After 24 hours, the conditioned medium is extracted. Migrationtests were conducted using PC3 tumor cells as a model and as achemoattractant medium: either culture medium containing 0% FCS(negative control), or culture medium containing 10% FCS (positivecontrol), or conditioned medium of human medullary adipocytes (CM-MAd).The level of migrant cells in the presence of CM-MAd is similar to thatobtained in the presence of medium containing 10% FCS, confirming theexistence of targeted migration induced by CM-MAd and thus the presenceof chemokines in these medullary adipocyte secretions.

FIG. 9B: Using an ELISA test, the presence of the chemokine CCL7 wasdemonstrated in CM-MAd in a quantity four times greater than thatpresent in the conditioned medium of human subcutaneous adipose AT andtwo times greater than that of human periprostatic adipose tissue.

FIGS. 9C and 9D: The chemokine (CCL7) and the receptor (CCR3) thereofplay a role in the targeted migration of PC3 tumor cells against CM-MAd,since this migration is inhibited by almost 50% using a CCR1/CCR3inhibitor (UCB35625) and by almost one third using blocking antibodiestargeted against CCR3 and CCL7, whereas antibodies targeted against CCR3are ineffective.

FIGS. 9E and 9F: In the case of obesity, there is a strengthening of theCCL7-CCR3 pathway. Firstly, the secretion of CCL7 by medullaryadipocytes, assayed by ELISA, is almost doubled in obese patients(BMI>30 mg/m²) compared to non-obese patients (BMI<25 mg/m²). The levelof secretion of CCL7 in medullary adipocytes is much greater inmedullary adipocytes than subcutaneous adipocytes (including in obesesubjects). Moreover, the secretions from medullary adipocytes of obesepatients are more chemoattractant than those from non-obese patients andinduce approximately 50% additional migration. Interestingly, using theinhibitor of CCR1 and CCR3 (UCB35625), the increase in migrationassociated with obesity is almost completely reversed.

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1-4. (canceled)
 5. An ex vivo method for determining the degree ofaggressiveness of a prostate cancer tumor in a human subject sufferingfrom prostate cancer, or for determining the risk of biologicalrecurrence of prostate cancer in a subject, comprising the followingsteps: (a) determining the concentration or level of expression of thechemokine receptor type 3 (CCR3) in a sample of prostate tumor cellsobtained from said subject, (b) comparing the concentration or level ofexpression of the CCR3 determined in step (a) with the referenceconcentration or level of expression of the CCR3 in healthy prostateepithelium, wherein a concentration or a level of expression of CCR3, insaid sample of prostate tumor cells from said subject, greater than saidreference concentration or level of expression is an indication of anaggressive prostate cancer tumor with a high extraprostaticdissemination potential or of a risk of biological recurrence ofprostate cancer.
 6. The method according to claim 5, wherein themeasurement of the concentration of CCR3 in a sample of prostate cellsand/or of prostate epithelium cells is performed by immunoassaycomprising a specific antibody for CCR3.
 7. The method according toclaim 5, wherein said human subject is an adult male who is obese and/orhas abundant PPAT (periprostatic adipose tissue).
 8. (canceled)
 9. Amethod of treating extension of prostate cancer outside the prostaticcapsule in a human subject comprising administering to the human subjectany of: (a) an inhibitor of CCL7/CCR3 interaction selected from thegroup consisting of an organic molecule, a peptide, an antibody againstCCR3 or CCL7, a peptidomimetic recognizing CCR3 or CCL7, and an aptamerrecognizing CCL7 or CCR3, (b) an inhibitor of CCR3 receptor expressiontargeting the gene encoding CCL7 or the gene encoding CCR3 selected fromthe group consisting of an antisense oligonucleotide, interfering RNA,small hairpin RNA (shRNA) and a ribozyme.
 10. The method according toclaim 9, wherein the CCR3 inhibitor is an CCR3 antagonist.
 11. Themethod according to claim 9, wherein the organic molecule is a compoundselected from any of group consisting of compounds having formulas I toDCCLXXXIV.
 11. The method of claim 9, wherein the organic molecule is acompound having the formula DLI.